Luciferase and methods for measuring intracellular ATP using the same

ABSTRACT

The present invention relates to luciferase having resistance to a surfactant and a method for measuring intracellular ATP which is characterized in that the luciferase having resistance to a surfactant is used in this method comprising the steps of: a first step wherein ATP is extracted from cells in a sample; a second step wherein light emission is produced by adding a luminescence reagent containing luciferase to the extracted ATP solution; and a third step wherein the light emission is measured.

TECHNICAL FIELD

[0001] The present invention relates to novel luciferase havingresistance to a surfactant and a method for measuring intracellular ATPusing the same.

BACKGROUND ART

[0002] Intracellular ATP is routinely measured for determining thepresence of cells in a sample or the number of cells in the fields offood sanitation, biology, clinical examinations, medical science,ultrapure water, and environmental science. A general method formeasuring intracellular ATP comprises the steps of adding an ATPextraction reagent containing a surfactant as an effective component toa sample containing cells, extracting intracellular ATP, adding aluminescence reagent containing luciferase into the sample, and thenmeasuring the total amount of light emitted.

[0003] Luciferase is an enzyme that catalyzes luminescence reaction ofluciferin, which is a substrate, in the presence of ATP and magnesiumion. Luciferase used in a method for measuring intracellular ATPincludes those derived from firefly species, such as GENJI firefly(Luciola cruciata), HEIKE firefly (Luciola lateralis), North Americanfirefly and Russian firefly, etc.

[0004] Intracellular ATP can be extracted by adding an ATP extractionreagent to a sample containing cells and then stirring the sample.

[0005] To make full use of the capabilities of the extraction reagent,preferably the reaction agent is added so that the concentration of asurfactant becomes 0.05% or more of the mixture of the sample and theextraction reagent. However, a condition where the concentration of thesurfactant is 0.05% or more, this inhibits significantly the enzymereaction in the process of measuring ATP concentration bybioluminescence. Thus the sensitivity and accuracy of measurement arelargely impaired. This is because a surfactant at such a highconcentration lowers luciferase activity.

[0006] For example, North American firefly luciferase activity decreasesto about 20% in the presence of 0.1% benzalkonium chloride (See Table1).

[0007] On the other hand, inhibition of the bioluminescent reaction canbe reduced with a lower concentration of surfactant. However, in thiscase the extraction efficiency for ATP would be insufficient.

[0008] A method wherein cyclodextrin or its derivative is used is aknown method for suppressing the inhibition of luminescence reaction bya surfactant (Japanese Patent Application Laid-Open No. 6-504200).

[0009] Among methods for measuring intracellular ATP whereinintracellular ATP is extracted by allowing a sample to contact with asurfactant and subsequently ATP is measured by luciferin-luciferasebioluminescent reaction method, a method for measuring intracellular ATPcharacterized by the application of the bioluminescent reaction methodafter allowing a sample, from which ATP is extracted, to contact withcyclodextrin (Japanese Patent Application Laid-Open Publication No.7-203995) is also known.

[0010] There has been no attempt so far to suppress the inhibition ofbioluminescent reaction due to a surfactant focusing on luciferase.

[0011] The purpose of the invention is to provide a novel luciferasehaving anti-surfactant resistance, whose activity is not impaired by thepresence of a surfactant at a high concentration. The other purpose ofthe invention is to provide a method, comprising the steps of extractingintracellular ATP using a surfactant and measuring intracellular ATP bybioluminescent reaction using a luciferase, which can lower theinhibition of bioluminescent reaction due to a surfactant without adecrease in efficiency in extracting intracellular ATP.

[0012] In the context of this Specification, the term “suppress” is usedto describe significant reduction of the inhibition of the luminescencereaction by a surfactant and the complete elimination of thisinhibition.

DISCLOSURE OF THE INVENTION

[0013] The present invention relates to a luciferase havinganti-surfactant resistance.

[0014] The luciferase having resistance to a surfactant includes aluciferase, wherein an amino acid at the 490-position, or an amino acidcorresponding to the amino acid at 490-position of GENJI firefly orHEIKE firefly is substituted by an amino acid other than glutamic acid,e.g., lysine, in the amino acid sequence of a wild-type fireflyluciferase.

[0015] Further, the luciferase having resistance to a surfactantincludes a polypeptide consisting of (a) or (b):

[0016] (a) A polypeptide consisting of the amino acid sequence shown inSEQ ID NO:4,

[0017] (b) A polypeptide comprising additions, deletions, orsubstitutions of one or more of amino acids in the polypeptide of (a),and having luciferase activity resistant to a surfactant, or

[0018] a polypeptide consisting of (a) or (b):

[0019] (a) A protein consisting of an amino acid sequence shown in SEQID NO:6,

[0020] (b) A protein comprising additions, deletions, or substitutionsof one or more of amino acids in the polypeptide of (a), and havingluciferase activity resistant to a surfactant.

[0021] Further, the present invention relates to a luceferase geneencoding the luciferase having resistance to a surfactant.

[0022] Furthermore, the present invention relates to a recombinantvector containing the luceiferase gene encoding the luciferase havingresistance to a surfactant.

[0023] The present invention also relates to a transformant containingthe recombinant vector.

[0024] In addition, the present invention relates to a method forproducing the luciferase, comprising the steps of culturing therecombinant in a medium, and collecting luciferase with resistance to asurfactant from the culture product.

[0025] Moreover the present invention relates to a method for measuringintracellular ATP, comprising the steps of a first step wherein ATP isextracted in the presence of a surfactant from cells in a sample; asecond step wherein a luminescence reagent containing luciferase isadded to the extracted ATP solution so as to cause light emission; and athird step wherein the light emission is measured, and characterized inthat luciferase having resistance to a surfactant is used.

[0026] This specification encompasses the description and/or drawingsgiven in Japanese Patent Application No. H09-361022.

BRIEF DESCRIPTION OF DRAWINGS

[0027]FIG. 1 shows a production processes for a mutant luciferase HIK.

[0028]FIG. 2 shows change with time of light emission from natural typeluciferase.

[0029]FIG. 3 shows a comparative resistance against benzalkoniumchloride of mutant luciferase.

[0030]FIG. 4 shows a comparative resistance against benzetonium chlorideof mutant luciferase.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention will now be described in detail.

[0032] [Luciferase Having Resistance to Surfactant]

[0033] Luciferase having resistance to a surfactant according to thepresent invention is as described below.

[0034] The term “having resistance to a surfactant” corresponds to anyone of the following features.

[0035] (1) When compared to known luciferase, the luciferase of thepresent invention leads to an increased initial amount of light emittedin the presence of a surfactant. Here the term “compare” means, forexample, where the luciferase of the present invention is produced byintroducing mutation into an amino acid sequence of known luciferase, tocompare light emission from luciferase before and after the introductionof a mutation.

[0036] (2) When compared to known luciferase, the luciferase of thepresent invention shows a gentle decrease in its activity in thepresence of a surfactant.

[0037] (3) The luciferase of the present invention has the remainingactivity of more than 85% in the present of 0.4% surfactant.

[0038] Hereinafter “luciferase having resistance to a surfactant” isreferred to as “surfactant-resistant luciferase.”

[0039] The term “activity” means the catalytic activity ofbioluminescent reaction. Further any surfactant can be used in thepresent invention so far as it can be used in the measurement system forintracellular ATP. These surfactants include an anionic surfactant,cationic surfactant, ampholytic surfactant, non-ionic surfactant. Aspecific reagent is benzalkonium chloride or benzetonium chloridecontaining quaternary ammonium salt as a major component.

[0040] The luciferase of the present invention can be prepared fromluminescence organs of luminescent organisms. The luminescent organismsinclude luminescent insects and luminescent bacteria. The luminescentinsects include those belonging to the order Cleoptera, such as thosebelonging to the family firefly and the family Pyrophorus. Specificexamples include GENJI firefly, HEIKE firefly, North American firefly,Russian firefly, Pynophorus plagiophthalamus, Arachnocampa luminosa, andRail worm. Further the luciferase of the present invention is obtainedby cloning a luciferase gene from the luminescent organism and allowingthe gene to express using an appropriate vector-host system.

[0041] Moreover, the luciferase of the present invention can be obtainedby introducing mutation such as additions, deletions, and substitutionsinto an amino acid sequence of well-known luciferase. Well-known geneticengineering techniques can be used to introduce mutation into an aminoacid sequence. In this case firstly, a mutation such as an addition,deletion, or substitution is introduced into a nucleotide sequence of aluciferase gene derived from the abovementioned luminescent organism ora well-known luciferase gene by genetic engineering techniques so as togenerate a mutant luciferase gene. Subsequently, the mutant gene isincorporated into an appropriate host-vector system, thereby generatinga recombinant microorganism. Then the recombinant microorganismsproducing the luciferase of the present invention are selected byscreening. The selected recombinant microorganisms are cultured in amedium. Finally the luciferase can be collected from the cultureproduct.

[0042] Hereinafter surfactant-resistant luciferase obtained byintroduction of a mutation into an amino acid sequence is referred to as“mutant luciferase.”

[0043] The mutant luciferase is for example, luciferase wherein an aminoacid corresponding to an amino acid at the 490-position of the GENJIfirefly luciferase or the HEIKE firefly luciferase, is substituted by anamino acid other than glutamic acid in an amino acid sequence of awild-type firefly luciferase. The amino acid other than glutamic acid isa basic amino acid. Specific examples include lysine, arginine, andhistidine. The term “an amino acid corresponding to the amino acid atthe 490-position of the GENJI or the HEIKE firefly luciferase” means anamino acid corresponding to the amino acid at the 490-position of theGENJI or HEIKE firefly luciferase when the determined amino acidsequence of luciferase is compared to an amino acid sequence of theGENJI or HEIKE firefly luciferase.

[0044] Moreover, in the GENJI or HEIKE firefly luciferase, the aminoacid at the 490-position is glutamic acid. Further, in North Americanfirefly luciferase, “an amino acid corresponding to the amino acid atthe 490-position of the GENJI or the HEIKE firefly luciferase”corresponds to the glutamic acid at the 487-position.

[0045] More specifically, the mutant luciferase is a polypeptidecomprising an amino acid sequence shown in SEQ ID NO:1 or 2, or saidamino acid sequence wherein one or more amino acids are added, deletedor substituted.

[0046] [Method for Producing Mutant Luciferase by Genetic EngineeringTechniques]

[0047] A method for generating mutant luciferase by genetic engineeringtechniques will now be described as follows.

[0048] The mutant luciferase is produced by introducing mutation such asadditions, deletions, and substitutions into a nucleotide sequence ofknown luciferase and allowing an appropriate vector-host system toexpress the gene.

[0049] The known luciferase genes includes, but are not limited to, afirefly luciferase gene, more specifically a wild-type HEIKE fireflyluciferase gene (Japanese Patent Application Laid-Open No. 2-171189) anda thermostable HEIKE firefly luciferase gene (Japanese PatentApplication Laid-Open No. 5-244942).

[0050] i) A method for introducing mutation into a luciferase gene is,for example a method wherein the gene and a mutagen are allowed tocontact with each other. Specific examples of the mutagen includehydroxylamine, nitrous acid, sulfurous acid, and 5-bromouracil. Further,ultra violet irradiation, cassette mutagenesis, and site-directedmutagenesis using PCR can also be used. Furthermore, a mutant lucefirasegene having a mutation at a desired position can be generated byannealing chemically synthesized DNA.

[0051] ii) Next, the mutant luciferase gene is inserted into a vectorDNA having such as a promoter sequence, a marker gene, and a replicationorigin, etc, thereby producing a recombinant plasmid. Any vector DNA canbe used so far as it can be replicated in a host cell. Examples of thevector DNA include plasmid DNA and bacteriophage DNA. When the host cellis Escherichia coli, examples of the vector DNA include plasmid pUC119(Takara Shuzo Co., Ltd.), pBluescript SK+(Stratagene), pMAL-C2 (NEWEngland Labs), pGEX-5X-1 (Pharmacia), pXal (Boehringer), and pMA56(G.Ammerer, Meth. Enzymol., 101, 192, 1983).

[0052] iii) Subsequently, an appropriate host cell is transformed ortransduced with the above recombinant plasmid, and screening isperformed for recombinant microorganisms having the ability to producethe mutant luciferase.

[0053] Any host cells including eucaryotic and prokaryotic cells can beused. The eucaryotic cells include animal, plant, insect, yeast cells.The prokaryotic cells include Escherichia coli, Bacillus subtilis, andActinomyces. The animal cells include CHO, COS, HeLa cells and cells ofmyeloma cell lines. The prokaryotic cells include microorganisms belongto the genus Escherichia, such as Escherichia coli JM101 (ATCC 33876),JM109 (produced by Takara Shuzo Co., Ltd.), XL1-Blue (produced byStratagene), and HB101 (ATCC33694).

[0054] Transformation in the present invention can be performed by forexample, D. M. Morrison's method (Meth. Enzymol., 68, 326-331, 1979);Transduction can be conducted by for example, B. Hohn's method (Meth.Enzymol., 68, 299-309, 1979). Methods for purification of recombinantDNA from recombinant microorganisms include P. Guerry's method(J.Bacteriology, 116, 1064-1066, 1973), and D. B. Clewell's method(J.Bacteriology, 110, 667-676, 1972).

[0055] The nucleotide sequence of a gene inserted into the recombinantDNA can be determined by, for example Maxam-Gilbert method (Proc. Natl.Acad. Sci. USA, 74, 560-564, 1977), and Dideoxy method (Proc. Natl.Acad. Sci. USA, 74, 5463-5467, 1977).

[0056] iv) The mutant luciferase of the present invention can beproduced by culturing the recombinant microorganisms obtained in themanner described above in media.

[0057] When the host cell is Escherichia coli, recombinant E.coli may becultured by solid culture methods, preferably liquid culture methods.

[0058] A culture medium of the present invention contains one or morenitrogen sources, such as yeast extract, tryptone, peptone, meatextract, corn steep liquor or exudate of soy bean or wheat bran, towhich one or more of inorganic salts, such as sodium chloride, potassiumphosphate, dipotassium phosphate, magnesium sulfate, magnesium chloride,ferric chloride, ferric sulfate or manganese sulfate are added. Ifnecessary sugar and vitamins are added to this medium. Further theinitial pH of the medium is preferably adjusted within pH 7 to 9.Moreover the culture is performed at a temperature within 30° C. to 42°C., preferably at around 37° C. for 3 to 24 hours, preferably for 5 to 8hours. Preferable culture methods include aeration-agitation submergedculture, shaking culture, and static culture.

[0059] To recover mutant luciferase from the culture product after thecompletion of culturing recombinant E.coli, standard means forcollecting enzymes can be employed. That is, the culture product iscentrifuged to obtain cells. Then the cells are disrupted by treatmentwith lytic enzymes, such as lysozyme, ultrasonication, or milling. Fusedprotein is discharged out of the cell. Subsequently insoluble substancesare removed by filtration or centrifugation, so that a crude enzymesolution containing mutant luciferase can be obtained.

[0060] In the present invention the above crude enzyme solution can beused as authetic protein matter, or alternatively it can further bepurified to higher purity by standard protein purification techniques.These techniques including sulfate salting out, organic solventprecipitation, ion exchange chromatography, hydrophobic chromatography,gel filtration chromatography, adsorption chromatography, affinitychromatography, and electrophoresis can be used solely or incombination.

[0061] The use of surfactant-resistant luciferase of the presentinvention allows the addition of a surfactant at a high concentration inthe extraction process for intracellular ATP.

[0062] [Detection of Intracellular ATP of the Present Invention]

[0063] Detection of intracellular ATP of the present invention will bedescribed as follows.

[0064] i) First, ATP extraction reagent containing surfactant as aneffective component is added to a sample containing cells so as toextract intracellular ATP out of the cells. The term “cells” refers tothe cells derived from animal, plant, microorganism (e.g., yeasts, mold,fungi, bacteria, actinomyces, unicellular algae, viruses, and protozoa).

[0065] Any sample can be used so far as it contains the above cells.These samples include, but are not limited to, foods and drinks,pharmaceuticals, cosmetics, seawater, river water, industrial water,sewage, soil, urine, feces, blood, sputum, pus, and culture product ofthe above cells. A sample solution can also be prepared by suspendingthese samples in an appropriate solvent, such as distilled water,physiological saline, phosphoric acid buffer, Tris buffer, or sodiumacetate buffer. When a fluid specimen contains solids, the fluidspecimen is suspended in an appropriate solvent or homogenized using amixer so that it can be handled in the same manner as that in liquidform.

[0066] A sample of a filter membrane can also be prepared by filteringthe above sample in liquid form through a hydrophilic or hydrophobicfilter membrane. The hydrophilic or hydrophobic filter membrane by whichcells are captured can be used as a sample. In such a case, a film- orsheet-type hydrophilic filter membrane made of hydrophilicpolytetrafluoroethylene, hydrophilic polyvinylidenefluoride, hydrophilicpolyamide, acetylcellulose, and nitrocellulose, etc., can be used.Hydrophobic filter membranes made of PVDF (polyvinylidenefluoride), PTFE(polytorafluoroethylene), and PE (polyethylene) etc., can be used.

[0067] Surfactants include anionic surfactants, cationic surfactants,ampholytic surfactants, and non-ionic surfactants. Anionic sulfactantsinclude sodium dodecyl sulfate (SDS), lauryl potassium sulfate, sodiummonolauroyl phosphate, and sodium alkylbenzenesulfonic acid. Cationicsurfactants include benzalkonium chloride (BAC), benzetonium chloride(BZC), cetylpyridinium chloride, cethyltrimethylammonium bromide, andmyristyldimethylbenzylammonium chloride. ampholytic surfactants includeTwittergent Detergent 3-08, 3-10, 3-12, 3-14, 3-16, and Tego. Finallynon-ionic surfactants include Tween 20, 60, and 80, Span 60 and 80,Triton X-45 and x-100, polyoxyethylene ether, and polyoxyethylene laurylether.

[0068] Any concentration of a surfactant can be employed so far as itallows full expression of the ability to extract ATP. Preferableconcentration of a surfactant is 0.05% or more of the mixture of asample and ATP extraction reagent.

[0069] A sample and ATP extraction reagent are contacted with from eachother at room temperature or with heating.

[0070] ii) After ATP extraction, bioluminescent reagent is added to thesample containing surfactant-resistant luciferase so as to causeemission. Then the light emission is measured.

[0071] When surfactant-resistant luciferase is derived from a firefly,the bioluminescent reagents are those containing e.g., the followingcomponents (a) to (c).

[0072] (a) surfactant-resistant luciferase

[0073] (b) luciferin

[0074] (c) magnesium ions or other metal ions

[0075] Further in addition to the above components, substances involvingpH preparation or improved shelf life may be added. Such substancesinclude EDTA 2Na, dithiothreitol, ammonium sulfate, sucrose,2-mercaptoethanol, HEPES, Tricine, and Tris.

[0076] iii) The amount of light emitted by the addition of abioluminescent reagent can be measured by a luminometer such as alumitester K-100 produced by Kikkoman Corporation, a luminescence readerBLR-201 produced by Aloka Co.,Ltd. (an improved type, or a Lumat LB9501produced by Berthold. When a filter membrane by which cells are capturedis used as a sample, the cells can be counted using a bioluminescentimage analysis system device to photograph spots on the filter membrane.Such a device is ARGUS-50/CL (with taper fiber: produced by HamamatsuPhotonics K.K.).

[0077] The present invention will now be described in detail by the useof examples. However the technical field of the present invention is notlimited by these examples.

EXAMPLE 1 Surfactant Resistance of Natural Type Luciferase Derived fromVarious Fire Fly Species

[0078] (Method of Preparing Wild Type Luciferase Derived from VariousFirefly Species)

[0079] Luciferase derived from GENJI and HEIKE fireflies was preparedaccording to the following methods. 1 mM ethylenediamine-4-acetate-2-sodium and 2mM phenylmethylsulfonylfluoride wereadded to 25mM Tris (hydroxy) aminomethane-hydrochloric acid buffer.Further ammonium sulfate was added to this solution so as to achieve 10%saturation. Tail portions of the various firefly species were added tothis mixture at pH 7.8, and then disrupted using Hiskotoron (produced byNichionrikakikaiseisakusho). The resulting solution was centrifuged at12,000 r.p.m. for 20 minutes to obtain supernatants as startingmaterials for purification. The purification was conducted by theprocess comprising salting out of ammonium sulfate, Ultrogel Ac A34(produced by LKB) column, and hydroxy-apatite HPLC (produced byTOSHOH,TSK gel HA-1000) column. Finally an electrophoretically homogenoussample was obtained. In addition the luciferase derived from NorthAmerican firefly is a commercial product (Sigma, L-9506).

[0080] (Method of Determining Luciferase Activity)

[0081] A luciferase sample was properly diluted using enzyme-dilutedsolution (1 mM EDTA, 1 mM 2-mercaptoethanol, 1% BSA, 50 mM HEPES,(pH7.5)). To 100 μl of this solution, 100 μl of substrate solution (1.4mM luciferin, 40 mM ATP, 300 mM MgSO₄. 7H₂O, 50 mM HEPES, (pH 7.5)) wasadded.

[0082] The light emission was measured using BLE-201 Luminescence reader(produced by Aloka Co., Ltd.) under the following conditions.

[0083] Measuring range: ×100

[0084] Numerical value displayed: ×1000

[0085] Measuring temperature: 30° C.

[0086] Measuring time: 20 seconds

[0087] 1 MLU (mega light unit) /ml is a value for activity when themeasured value under these conditions was 1 Kcount.

[0088] (Method of Determining Surfactant-Resistance)

[0089] Enzyme samples were obtained by preparing luciferase samplesderived from various firefly species using enzyme-diluted solution (1 mMEDTA, 1 mM 2-mercaptoethanol, 5% glycerol, 50 mM HEPES, (pH7.5)) toachieve 0.5 MLU/ml concentration.

[0090] 50 μl of 0.4% benzalkonium chloride (25 mM Tricine at pH 7.75)and then 50 μl of the enzyme sample were added to 100 μl of substratesolution (4 mM ATP, 0.4 mM luciferin, 10 mM magnesium sulfate, 50 mMHEPES (pH 7.5)). After the solution was stirred for 5 seconds, the lightemission was measured every second using Berthold Lumat LB-9501 for 1minute.

[0091]FIG. 2 shows the results. Along the vertical axis in this figure,the relative ratio of the light emission was plotted with the initialamount of light emitted considered to be 100% upon use of 25mM Tricine(pH 7.75) instead of 0.4% benzalkonium chloride.

[0092] As shown in these results, North American firefly luciferase waslow in the initial light emission and the light emission decayedrapidly. This was caused by the low surfactant-resistance of the NorthAmerican firefly luciferase. This can lead to low sensitivity andaccuracy in measuring such values. On the other hand, GENJI fireflyluciferase showed an initial light emission higher than that of NorthAmerican firefly luciferase. That is, GENJI firefly luciferase was shownto have a surfactant resistance superior to that of North Americanfirefly luciferase. Furthermore, HEIKE firefly luciferase showed aninitial light emission higher than that of GENJI firefly luciferase andthe emission decayed slowly. Therefore, HEIKE firefly luciferase hasgood surfactant resistance, superior to that of GENJI fireflyluciferase. These results suggest that the degree of surfactantresistance of luciferase varies according to the firefly species.

EXAMPLE 2 Preparation of Mutant Luciferase HLK and HIK

[0093] Two types of mutant luciferase (named “HLK” and “HIK”) wereprepared according to the following methods.

[0094] (Production of a Gene Encoding Mutant Luciferase HLK)

[0095] A mutant luciferase gene was produced by site-directedmutagenesis using PCR. A plasmid pHLf7-217Leu described in JapanesePatent Application Laid-Open No. 5-244942 was used as a template for PCRreaction. The pHLf7-217Leu was a recombinant plasmid prepared byinserting a thermostable HEIKE firefly luciferase gene, in which anamino acid corresponding to Ala at the 217-position was substituted fora Leu-encoding gene, into a plasmid pUC119. In addition, E. coli JM101,to which the recombinant plasmid pHLf7-217Leu had been introduced, hasbeen named E.coli JM101 (pHLf7-217Leu) and was deposited on Apr. 22,1992 as FERM BP-3840 with the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology (1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan).

[0096] The primer for PCR reaction was an oligonuceltide having anucleotide sequence shown in SEQ ID No: 1 or 2. The DNA polymerase was aKOD dash polymerase (produced by TOYOBO). A PCR reaction cycle (94° C.for 30 seconds, 50° C. for 2 seconds, and 74° C. for 3 minutes) wasrepeated for 30 times according to the examples attached to KOD dashpolymerase. The PCR product was ligated into a circular recombinantplasmid pHLfLK using standard techniques.

[0097] Sequencing of a mutant luciferase gene contained in the pHLfLKwas performed. Reaction was conducted using a Diprimer Taq SequencingKit (produced by Applied Biosystems). Then the eletrophoretic analysiswas performed using ABI 373A DNA sequencer (produced by AppliedBiosystems). The entire nucleotide sequence of the obtained mutantluciferase gene is shown in SEQ ID NO: 3, and the amino acid sequence ofa polypeptide encoded by this gene is shown in SEQ ID NO: 4. In themutant luciferase gene, the genetic portion corresponding to alanine atthe 217-position of wild-type HEIKE firefly luciferase was substitutedby a gene encoding leucine, the genetic portion corresponding toglutamic acid at the 490-position of the same was substituted by a geneencoding lysine. The pHLfLK-introduced E.coli. JM109 strain was namedE.coli JM109 (pHLfLK) (see FIG. 1). E. coli JM109 (pHLfLK) was depositedas FERM BP-6147 on Oct. 16, 1997) with the National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, Japan.

[0098] The polypeptide shown in SEQ ID NO:4 was named the mutantluciferase HLK.

[0099] (Preparation of Gene Encoding Mutant Luciferase HIK)

[0100] A mutant luciferase gene was prepared using the plasmidpHLf7-217Ile described in Japanese Patent Application Laid-Open No.5-244942. The plasmid pHLf7-217Ile was a recombinant plasmid prepared byinserting a thermostable HEIKE firefly luciferase gene, in which anamino acid corresponding to Ala at the 217-position was substituted fora Ile-encoding gene, into a plasmid pUC119. The transformant strainobtained using this plasmid was deposited on Apr. 22, 1992 as FERMBP-3841 with the National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology, Japan.

[0101] About a 560 bp fragment obtained by cutting the pHLfLK with EcoRVand NarI was obtained by agarose gel electrophoresis. Then the fragmentwas inserted into the pHLf7-217Ile treated with the same restrictionenzymes.

[0102] The resulting recombinant plasmid has been named pHLfIK and theplasmid-introduced E.coli JM109 strain has been named E.coli JM109(pHLflK).

[0103]E.coli JM109 (pHLflK) was deposited on Oct. 16, 1997 as FERMBP-6146 with the National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology, Japan.

[0104] The entire nucleotide sequence of the mutant luciferase genecontained in the pHLflK is shown in SEQ ID NO: 5, and the amino acidsequence of a polypeptide encoded by this gene is shown in SEQ ID NO: 6.In the mutant luciferase gene, the genetic portion corresponding toalanine at the 217-position of wild-type HEIKE firefly luciferase wassubstituted by a gene encoding isoleucine, the genetic portioncorresponding to glutamic acid at the 490-position of the same wassubstituted by a gene encoding lysine (see FIG. 1).

[0105] A polypeptide shown in SEQ ID NO:6 was named the mutant HIKfirefly.

EXAMPLE 3 Preparation of Mutant Luciferase HLK and HIK

[0106]E.coli JM109 (pHLfLK) and E.coli JM109 (pHLfIK) were inoculated onLB media (1% Bacto-trypton (W/V), 0.5% yeast extract (W/V), 0.5% NaCl(W/V), ampicillin (50 μg/ml), 1.4% agar (W/V)), each containingampicillin, and cultured at 37° C. for 18 hours. The resulting culturefluid was centrifuged at 8000 r.p.m. for 10 minutes. The precipitatedcells were suspended in 0.1M potassium phosphate buffer at pH 7.8 (0.1Mammonium sulfate, 1 mM EDTA) were disrupted by ultrasonication.

[0107] Next, crude enzyme solution was obtained by centrifugation at12000 r.p.m. for 10 minutes. The obtained enzyme solution was purifiedusing the above purification techniques such that it becomes anelectrophoretically homogenous sample.

EXAMPLE 4 Surfactant Resistance of Mutant Luciferase HLK and HIK

[0108] (Changes in Emission with Time)

[0109] To compare surfactant resistance of mutant luciferase with thatof known luciferase, changes in emission with time were measuredaccording to the aforementioned methods of measuring surfactantresistance. FIG. 3 shows the results obtained by the use of 0.4%benzalkonium chloride (25 mM Tricine (pH 7.75)). FIG. 4 shows theresults obtained by the use of 0.8% benzethonium chloride (25 mM Tricine(pH 7.75)).

[0110] “HEIKE I mutant” in this figure is thermostable HEIKE fireflyluciferase (described in Japanese Patent Application Laid-Open No.5-244942) wherein Ala at the 217-position of wild-type HEIKE fireflyluciferase is substituted for Ile. “HEIKE L mutant” is thermostableHEIKE firefly luciferase (Japanese Patent Application Laid-Open No.5-244942) wherein Ala at the 217 position of wild-type HEIKE luciferaseis substituted by Leu. “HIK” is a mutant wherein Glu at the 490-positionof HEIKE I mutant is substituted by Lys, that is, the mutant luciferaseHIK prepared in Example 3. “HLK” is a mutant wherein Glu at the490-position of HEIKE L mutant is substituted by Lys, that is, themutant luciferase HLK prepared in Example 3.

[0111] As can be seen in FIG. 3 which shows the results for benzalkoniumchloride, the emission of HIK decayed more slowly than that of the HEIKEI mutant. Comparison of HLK and HEIKE L mutant reveals that HLK hadinitial light emission improved by about 20%, and slower decay in thelight emission.

[0112] Therefore, the substitution of an amino acid at the 490-positionresulted in improved surfactant-resistance of a luciferase.

[0113] As shown in FIG. 4 which shows the results obtained by the use ofbenzethonium chloride, HIK showed decay in emission more slowly thanthat of HEIKE I mutant. Further HLK showed slower decay in lightemission than that of HEIKE L mutant. Therefore, the substitution of anamino acid at the 490-position resulted in improved surfactantresistance.

[0114] (Comparison of Emission Rate)

[0115] The influence of the enzyme solution, substrate solution andbenzalkonium chloride used when measuring change with time, on themeasurement values taken under actual emission measurement conditions,was examined. Table 1 shows the light emission measured using BertholdLumat LB-9501 under measuring conditions (5 seconds of waiting time, 3seconds of measuring time).

[0116] In addition, the emission rate (remaining activity) wascalculated by dividing the light emission measured in the presence of0.4% benzalkonium chloride by a control value. Here the control valuewas the light emission upon use of 25 mM Tricine at pH 7.75 instead of0.4% benzalkonium chloride. TABLE 1 Light emission (RLU) Withoutextraction With extraction Emission Luciferase type reagent reagent rate(%) North American firefly 452563 97790 21.6 GENJI firefly 409406 16780541.0 HEIKE firefly 425792 324724 76.3 HEIKE I mutant 422269 341039 80.8HEIKE L mutant 423728 343634 81.1 HIK 386429 345159 89.3 HLK 390289396764 101.7

[0117] North American firefly luciferase showed an emission rate as lowas 21.6%, suggesting a large decrease in sensitivity. On the other hand,the emission rates for GENJI and HEIKE firefly luciferase were 41.0% and76.3%, respectively, suggesting that the sensitivity of these fireflyluciferases were less affected than that of North American fireflyluciferase.

[0118] The emission rate for mutant luciferase HIK and HLK were 89.3%and 101.7%, respectively. These rates were far greater than those ofwild-type HEIKE firefly luciferase and thermostable HEIKE fireflyluciferase. Particularly the emission rate of HLK was almost 100%. Thatis, HLK can yield the same light emission regardless of the presence orabsence of a surfactant. Therefore, the sensitivity of HLK is totallyunaffected by the use of a surfactant, allowing measurement with highaccuracy.

[0119] (Comparison of IC50)

[0120] Benzalkonium chloride and various luciferases were contacted witheach other for 10 minutes. Then the benzalkonium chloride concentration(IC50), at which activity is inactivated by 50% was determined. Equalamounts of luciferase solution prepared at this concentration and 0.01to 0.1% benzalkonium chloride were mixed, and then allowed to stand for10 minutes at room temperature. Subsequently, 100 μl of substratesolution was added to the mixture. Immediately after addition, the lightemission was measured using Berthold Lumat LB-9501. IC50s obtained wereas shown in Table 2. TABLE 2 IC₅₀ for various luciferase Luciferase typeIC₅₀ (%) North American firefly 0.014 GENJI firefly luciferase 0.016HEIKE firefly luciferase 0.026 HEIKE I mutant 0.028 HEIKE L mutant 0.028HIK 0.032 HLK 0.035

[0121] North American firefly luciferase showed the lowest IC₅₀ amongthe three types of wild-type luciferase. That is, North American fireflyluciferase was shown to have the lowest resistance to a surfactant.HEIKE firefly luciferase showed the highest IC₅₀ among the wild-typeluciferase. HLK and HIK showed IC₅₀ higher than those of wild-type HEIKEfirefly luciferase and thermostable HEIKE firefly luciferase, suggestingthat the resistance was improved by the substitution of an amino acid atthe 490-position. Especially HLK showed IC₅₀ higher than that of HIK,indicating that HLK possesses the best surfactant-resistance.

EXAMPLE 5 Method for Measuring Intracellular ATP

[0122] Next, a method for measuring intracellular ATP using thesurfactant-resistant luciferase of the present invention will bedescribed.

[0123] A standard technique used herein was TCA extraction methodwherein intracellular ATP is extracted using trichloroacetic acid (TCA)and the amount of ATP extracted is measured using luciferin-luciferaseluminescence reaction. TCA extraction method is excellent in extractionefficiency. Further in TCA extraction method no inhibition ofluminescence reaction is caused by TCA because emission is measuredafter the sample containing TCA is diluted 1:100. Because of thisdilution, however, TCA extraction method is complicated and can cause adecrease in the measuring sensitivity.

[0124] 1. Materials

[0125] (1) Surfactant

[0126] Benzalkonium chloride (BAC, Japanese Pharmacopoeia) was used. ATPextraction reagent was prepared by dissolving this surfactant at 0.25%concentration into 25 mM Tricine (pH 7.75).

[0127] (2) Microorganisms

[0128] Four strains, Escherichia coli (ATCC 25922), Staphylococcusaureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) andEnterococcus faecalis (ATCC 29212) were used.

[0129] (3) Preparation of Samples

[0130] In standard techniques, a sample, undiluted solution, wasprepared by culturing the prescribed microorganisms on a normal brothmedium (produced by Eiken chemical Co., Ltd.) at 35° C. overnight. Inthe method of the present invention, a sample diluted solution wasprepared by diluting an undiluted solution of the culture fluid to 1:100with sterile water.

[0131] (4) Luciferase

[0132] Surfactant-resistant luciferase of the present invention were HIKand HLK. Control surfactant-resistant luciferase types were knownluciferase (North American firefly luciferase, GENJI firefly luciferase,HEIKE firefly luciferase, HEIKE I mutant, and HEIKE L mutant).

[0133] (5) Luminescence Reagent

[0134] Luminescence reagent was prepared by adding various luciferase tosolution containing 0.15 mM luciferin, 6 mM EDTA, 15 mM magnesiumacetate, 0.2 mM dithiothreitol, 0.5% BSA and 25 mM HEPES (pH 7.75).

[0135] The amount of luciferase to be added was prepared such that thelight emission produced when 100 μl of 2×10⁻⁸ M ATP standard solutionwas added to 100 μl of the luminescence reagent would be the same amountof the light emission produced when a luminescence reagent attached toLuciferase LU (Kikkoman Corporation) was used.

[0136] 2. Method for Measuring Intracellular ATP

[0137] (1) Method of the Present Invention

[0138] ATP extraction reagent 100 μl was added to 100 μl of a sample.The solution was allowed to stand for 20 seconds at room temperature.Then 100 μl of the luminescence reagent was added to this solution.Immediately after addition, the light emission was measured using LumatLB-9501 produced by Berthold.

[0139] (2) Standard Technique

[0140] 10% trichloro acetate solution 100 μl was added to 100 μl of asample and the solution was allowed to stand for 1 minute. 25 mM Tricine(pH 7.75) 9.8 ml was added to this extract, and then the extract waswell stirred. 25 mM Tricine (pH 7.75) and 100 μl of a luminescencereagent attached to CheckLite LU (produced by Kikkoman Corporation) wereadded to 100 l of the sample. Immediately after addition, the lightemission was measured using Lumat LB-9501 produced by Berthold.

[0141] 3. Results

[0142] Tables 3 and 4 show the results. The relative ratio of the lightemissions obtained by the use of the luminescence reagents using variousluciferase types is also shown in these tables. Here the light emissionobtained by the standard technique (TCA extraction method) was definedas 100%. TABLE 3 Detection of intracellular ATP E. coli S. aureus ATCCATCC 25922 25923 Measured Measured value Relative value RelativeMeasuring method (RLU) ratio (%) (RLU) ratio (%) Standard technique132794 (100.0) 130220 (100.0) (TCA extraction method) North Americanfirefly 153 (0.1) 163 (0.1) GENJI firefly 463 (0.3) 659 (0.5) luciferaseHEIKE firefly 76082 (57.3) 74019 (56.8) luciferase HEIKE I mutant 47655(35.9) 50031 (38.4) HEIKE L mutant 46217 (34.8) 51243 (39.4) HIK 97073(73.1) 76533 (58.8) HLK 87981 (66.3) 72182 (55.4)

[0143] TABLE 4 Detection of intracellular ATP P. aeruginosa E. faecalisATCC ATCC 27853 29212 Measured Relative Measured Relative value ratiovalue ratio Measuring method (RLU) (%) (RLU) (%) Standard technique168141 (100.0) 12427 (100.0) (TCA extraction method) North Americanfirefly 553 (0.3) 113 (0.1) GENJI firefly luciferase 1503 (0.9) 163(1.3) HEIKE firefly 117096 (69.6) 8132 (65.4) luciferase HEIKE I mutant80455 (47.8) 4586 (36.9) HEIKE L mutant 81069 (48.2) 4762 (38.3) HIK131134 (78.0) 7914 (63.7) HLK 131815 (78.4) 7998 (64.4)

[0144] No emission was obrserved for the luminescence reagent containingNorth American firefly luciferase. GENJI firefly luciferase showed weakemission. This is because the luciferase itself was devitalized by thesurfactant. Therefore, it was shown that the surfactant at highconcentration such as was used in this examination cannot be used as anATP extraction reagent for the luciferase.

[0145] Unlike North American firefly luciferase and GENJI fireflyluciferase, HEIKE firefly luciferase showed emission 60 to 70% of thatin TCA extraction method. HEIKE firefly luciferase was shown to possesssurfactant-resistance higher than those of North American fireflyluciferase and GENJI firefly luciferase.

[0146] Light emissions from HEIKE L mutant, and HEIKE I mutant which isthermostable HEIKE firefly luciferase were each equivalent to around 40%of that in TCA extraction method, and largely lower than that ofwild-type HEIKE firefly luciferase.

[0147] Each of the light emission from HIK and HLK, which issurfactant-resistant luciferase of the present invention, respectivelywas more intense than that from wild-type HEIKE luciferase andthermostable luciferase. Further the light emission in this case wasequivalent to 60 to 80% of that in TCA extraction method.

[0148] HIK and HLK are mutants wherein Glu at the 490-position of HEIKEI and HEIKE L mutants are substituted for Lys, respectively. That is,the introduction of said mutation into the amino acid at the490-position improved resistance to a surfactant. The sensitivity of HIKand HLK is less affected by ATP extraction reagent even at such a highconcentration employed in this examination, suggesting the use of HIKand HLK enable highly accurate measurement.

[0149] Industrial Applicability

[0150] The use of a novel surfactant-resistant luciferase according tothe present invention for measuring intracellular ATP allows thedetection without a decrease in luciferase activity even in the presenceof a surfactant at a high concentration.

[0151] All publications, patents and patent applications cited hereinare incorporated herein by reference in their entirety.

[0152] Sequence Listing Free Text

[0153] SEQ ID NO:1: A synthetic DNA

[0154] SEQ ID NO:2: A synthetic DNA

1 8 1 23 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 1 tgttgtactt aagaaaggaaaat 23 2 23 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 2 acagctcccggaagctcacc agc 23 3 1644 DNA Luciola lateralis CDS (1)..(1644) 3 atg gaaaac atg gag aac gat gaa aat att gtg tat ggt cct gaa cca 48 Met Glu AsnMet Glu Asn Asp Glu Asn Ile Val Tyr Gly Pro Glu Pro 1 5 10 15 ttt taccct att gaa gag gga tct gct gga gca caa ttg cgc aag tat 96 Phe Tyr ProIle Glu Glu Gly Ser Ala Gly Ala Gln Leu Arg Lys Tyr 20 25 30 atg gat cgatat gca aaa ctt gga gca att gct ttt act aac gca ctt 144 Met Asp Arg TyrAla Lys Leu Gly Ala Ile Ala Phe Thr Asn Ala Leu 35 40 45 acc ggt gtc gattat acg tac gcc gaa tac tta gaa aaa tca tgc tgt 192 Thr Gly Val Asp TyrThr Tyr Ala Glu Tyr Leu Glu Lys Ser Cys Cys 50 55 60 cta gga gag gct ttaaag aat tat ggt ttg gtt gtt gat gga aga att 240 Leu Gly Glu Ala Leu LysAsn Tyr Gly Leu Val Val Asp Gly Arg Ile 65 70 75 80 gcg tta tgc agt gaaaac tgt gaa gaa ttc ttt att cct gta tta gcc 288 Ala Leu Cys Ser Glu AsnCys Glu Glu Phe Phe Ile Pro Val Leu Ala 85 90 95 ggt tta ttt ata ggt gtcggt gtg gct cca act aat gag att tac act 336 Gly Leu Phe Ile Gly Val GlyVal Ala Pro Thr Asn Glu Ile Tyr Thr 100 105 110 cta cgt gaa ttg gtt cacagt tta ggc atc tct aag cca aca att gta 384 Leu Arg Glu Leu Val His SerLeu Gly Ile Ser Lys Pro Thr Ile Val 115 120 125 ttt agt tct aaa aaa ggatta gat aaa gtt ata act gta caa aaa acg 432 Phe Ser Ser Lys Lys Gly LeuAsp Lys Val Ile Thr Val Gln Lys Thr 130 135 140 gta act gct att aaa accatt gtt ata ttg gac agc aaa gtg gat tat 480 Val Thr Ala Ile Lys Thr IleVal Ile Leu Asp Ser Lys Val Asp Tyr 145 150 155 160 aga ggt tat caa tccatg gac aac ttt att aaa aaa aac act cca caa 528 Arg Gly Tyr Gln Ser MetAsp Asn Phe Ile Lys Lys Asn Thr Pro Gln 165 170 175 ggt ttc aaa gga tcaagt ttt aaa act gta gaa gtt aac cgc aaa gaa 576 Gly Phe Lys Gly Ser SerPhe Lys Thr Val Glu Val Asn Arg Lys Glu 180 185 190 caa gtt gct ctt ataatg aac tct tcg ggt tca acc ggt ttg cca aaa 624 Gln Val Ala Leu Ile MetAsn Ser Ser Gly Ser Thr Gly Leu Pro Lys 195 200 205 ggt gtg caa ctt actcat gaa aat ttg gtc act aga ttt tct cac gct 672 Gly Val Gln Leu Thr HisGlu Asn Leu Val Thr Arg Phe Ser His Ala 210 215 220 aga gat cca att tatgga aac caa gtt tca cca ggc acg gct att tta 720 Arg Asp Pro Ile Tyr GlyAsn Gln Val Ser Pro Gly Thr Ala Ile Leu 225 230 235 240 act gta gta ccattc cat cat ggt ttt ggt atg ttt act act tta ggc 768 Thr Val Val Pro PheHis His Gly Phe Gly Met Phe Thr Thr Leu Gly 245 250 255 tat cta act tgtggt ttt cgt att gtc atg tta acg aaa ttt gac gaa 816 Tyr Leu Thr Cys GlyPhe Arg Ile Val Met Leu Thr Lys Phe Asp Glu 260 265 270 gag act ttt ttaaaa aca ctg caa gat tac aaa tgt tca agc gtt att 864 Glu Thr Phe Leu LysThr Leu Gln Asp Tyr Lys Cys Ser Ser Val Ile 275 280 285 ctt gta ccg actttg ttt gca att ctt aat aga agt gaa tta ctc gat 912 Leu Val Pro Thr LeuPhe Ala Ile Leu Asn Arg Ser Glu Leu Leu Asp 290 295 300 aaa tat gat ttatca aat tta gtt gaa att gca tct ggc gga gca cct 960 Lys Tyr Asp Leu SerAsn Leu Val Glu Ile Ala Ser Gly Gly Ala Pro 305 310 315 320 tta tct aaagaa att ggt gaa gct gtt gct aga cgt ttt aat tta ccg 1008 Leu Ser Lys GluIle Gly Glu Ala Val Ala Arg Arg Phe Asn Leu Pro 325 330 335 ggt gtt cgtcaa ggc tat ggt tta aca gaa aca acc tct gca att att 1056 Gly Val Arg GlnGly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Ile 340 345 350 atc aca ccggaa ggc gat gat aaa cca ggt gct tct ggc aaa gtt gtg 1104 Ile Thr Pro GluGly Asp Asp Lys Pro Gly Ala Ser Gly Lys Val Val 355 360 365 cca tta tttaaa gca aaa gtt atc gat ctt gat act aaa aaa act ttg 1152 Pro Leu Phe LysAla Lys Val Ile Asp Leu Asp Thr Lys Lys Thr Leu 370 375 380 ggc ccg aacaga cgt gga gaa gtt tgt gta aag ggt cct atg ctt atg 1200 Gly Pro Asn ArgArg Gly Glu Val Cys Val Lys Gly Pro Met Leu Met 385 390 395 400 aaa ggttat gta gat aat cca gaa gca aca aga gaa atc ata gat gaa 1248 Lys Gly TyrVal Asp Asn Pro Glu Ala Thr Arg Glu Ile Ile Asp Glu 405 410 415 gaa ggttgg ttg cac aca gga gat att ggg tat tac gat gaa gaa aaa 1296 Glu Gly TrpLeu His Thr Gly Asp Ile Gly Tyr Tyr Asp Glu Glu Lys 420 425 430 cat ttcttt atc gtg gat cgt ttg aag tct tta atc aaa tac aaa gga 1344 His Phe PheIle Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly 435 440 445 tat caagta cca cct gct gaa tta gaa tct gtt ctt ttg caa cat cca 1392 Tyr Gln ValPro Pro Ala Glu Leu Glu Ser Val Leu Leu Gln His Pro 450 455 460 aat attttt gat gcc ggc gtt gct ggc gtt cca gat cct ata gct ggt 1440 Asn Ile PheAsp Ala Gly Val Ala Gly Val Pro Asp Pro Ile Ala Gly 465 470 475 480 gagctt ccg gga gct gtt gtt gta ctt aag aaa gga aaa tct atg act 1488 Glu LeuPro Gly Ala Val Val Val Leu Lys Lys Gly Lys Ser Met Thr 485 490 495 gaaaaa gaa gta atg gat tac gtt gct agt caa gtt tca aat gca aaa 1536 Glu LysGlu Val Met Asp Tyr Val Ala Ser Gln Val Ser Asn Ala Lys 500 505 510 cgtttg cgt ggt ggt gtc cgt ttt gtg gac gaa gta cct aaa ggt ctc 1584 Arg LeuArg Gly Gly Val Arg Phe Val Asp Glu Val Pro Lys Gly Leu 515 520 525 actggt aaa att gac ggt aaa gca att aga gaa ata ctg aag aaa cca 1632 Thr GlyLys Ile Asp Gly Lys Ala Ile Arg Glu Ile Leu Lys Lys Pro 530 535 540 gttgct aag atg 1644 Val Ala Lys Met 545 4 548 PRT Luciola lateralis 4 MetGlu Asn Met Glu Asn Asp Glu Asn Ile Val Tyr Gly Pro Glu Pro 1 5 10 15Phe Tyr Pro Ile Glu Glu Gly Ser Ala Gly Ala Gln Leu Arg Lys Tyr 20 25 30Met Asp Arg Tyr Ala Lys Leu Gly Ala Ile Ala Phe Thr Asn Ala Leu 35 40 45Thr Gly Val Asp Tyr Thr Tyr Ala Glu Tyr Leu Glu Lys Ser Cys Cys 50 55 60Leu Gly Glu Ala Leu Lys Asn Tyr Gly Leu Val Val Asp Gly Arg Ile 65 70 7580 Ala Leu Cys Ser Glu Asn Cys Glu Glu Phe Phe Ile Pro Val Leu Ala 85 9095 Gly Leu Phe Ile Gly Val Gly Val Ala Pro Thr Asn Glu Ile Tyr Thr 100105 110 Leu Arg Glu Leu Val His Ser Leu Gly Ile Ser Lys Pro Thr Ile Val115 120 125 Phe Ser Ser Lys Lys Gly Leu Asp Lys Val Ile Thr Val Gln LysThr 130 135 140 Val Thr Ala Ile Lys Thr Ile Val Ile Leu Asp Ser Lys ValAsp Tyr 145 150 155 160 Arg Gly Tyr Gln Ser Met Asp Asn Phe Ile Lys LysAsn Thr Pro Gln 165 170 175 Gly Phe Lys Gly Ser Ser Phe Lys Thr Val GluVal Asn Arg Lys Glu 180 185 190 Gln Val Ala Leu Ile Met Asn Ser Ser GlySer Thr Gly Leu Pro Lys 195 200 205 Gly Val Gln Leu Thr His Glu Asn LeuVal Thr Arg Phe Ser His Ala 210 215 220 Arg Asp Pro Ile Tyr Gly Asn GlnVal Ser Pro Gly Thr Ala Ile Leu 225 230 235 240 Thr Val Val Pro Phe HisHis Gly Phe Gly Met Phe Thr Thr Leu Gly 245 250 255 Tyr Leu Thr Cys GlyPhe Arg Ile Val Met Leu Thr Lys Phe Asp Glu 260 265 270 Glu Thr Phe LeuLys Thr Leu Gln Asp Tyr Lys Cys Ser Ser Val Ile 275 280 285 Leu Val ProThr Leu Phe Ala Ile Leu Asn Arg Ser Glu Leu Leu Asp 290 295 300 Lys TyrAsp Leu Ser Asn Leu Val Glu Ile Ala Ser Gly Gly Ala Pro 305 310 315 320Leu Ser Lys Glu Ile Gly Glu Ala Val Ala Arg Arg Phe Asn Leu Pro 325 330335 Gly Val Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Ile 340345 350 Ile Thr Pro Glu Gly Asp Asp Lys Pro Gly Ala Ser Gly Lys Val Val355 360 365 Pro Leu Phe Lys Ala Lys Val Ile Asp Leu Asp Thr Lys Lys ThrLeu 370 375 380 Gly Pro Asn Arg Arg Gly Glu Val Cys Val Lys Gly Pro MetLeu Met 385 390 395 400 Lys Gly Tyr Val Asp Asn Pro Glu Ala Thr Arg GluIle Ile Asp Glu 405 410 415 Glu Gly Trp Leu His Thr Gly Asp Ile Gly TyrTyr Asp Glu Glu Lys 420 425 430 His Phe Phe Ile Val Asp Arg Leu Lys SerLeu Ile Lys Tyr Lys Gly 435 440 445 Tyr Gln Val Pro Pro Ala Glu Leu GluSer Val Leu Leu Gln His Pro 450 455 460 Asn Ile Phe Asp Ala Gly Val AlaGly Val Pro Asp Pro Ile Ala Gly 465 470 475 480 Glu Leu Pro Gly Ala ValVal Val Leu Lys Lys Gly Lys Ser Met Thr 485 490 495 Glu Lys Glu Val MetAsp Tyr Val Ala Ser Gln Val Ser Asn Ala Lys 500 505 510 Arg Leu Arg GlyGly Val Arg Phe Val Asp Glu Val Pro Lys Gly Leu 515 520 525 Thr Gly LysIle Asp Gly Lys Ala Ile Arg Glu Ile Leu Lys Lys Pro 530 535 540 Val AlaLys Met 545 5 1644 DNA Luciola lateralis CDS (1)..(1644) 5 atg gaa aacatg gag aac gat gaa aat att gtg tat ggt cct gaa cca 48 Met Glu Asn MetGlu Asn Asp Glu Asn Ile Val Tyr Gly Pro Glu Pro 1 5 10 15 ttt tac cctatt gaa gag gga tct gct gga gca caa ttg cgc aag tat 96 Phe Tyr Pro IleGlu Glu Gly Ser Ala Gly Ala Gln Leu Arg Lys Tyr 20 25 30 atg gat cga tatgca aaa ctt gga gca att gct ttt act aac gca ctt 144 Met Asp Arg Tyr AlaLys Leu Gly Ala Ile Ala Phe Thr Asn Ala Leu 35 40 45 acc ggt gtc gat tatacg tac gcc gaa tac tta gaa aaa tca tgc tgt 192 Thr Gly Val Asp Tyr ThrTyr Ala Glu Tyr Leu Glu Lys Ser Cys Cys 50 55 60 cta gga gag gct tta aagaat tat ggt ttg gtt gtt gat gga aga att 240 Leu Gly Glu Ala Leu Lys AsnTyr Gly Leu Val Val Asp Gly Arg Ile 65 70 75 80 gcg tta tgc agt gaa aactgt gaa gaa ttc ttt att cct gta tta gcc 288 Ala Leu Cys Ser Glu Asn CysGlu Glu Phe Phe Ile Pro Val Leu Ala 85 90 95 ggt tta ttt ata ggt gtc ggtgtg gct cca act aat gag att tac act 336 Gly Leu Phe Ile Gly Val Gly ValAla Pro Thr Asn Glu Ile Tyr Thr 100 105 110 cta cgt gaa ttg gtt cac agttta ggc atc tct aag cca aca att gta 384 Leu Arg Glu Leu Val His Ser LeuGly Ile Ser Lys Pro Thr Ile Val 115 120 125 ttt agt tct aaa aaa gga ttagat aaa gtt ata act gta caa aaa acg 432 Phe Ser Ser Lys Lys Gly Leu AspLys Val Ile Thr Val Gln Lys Thr 130 135 140 gta act gct att aaa acc attgtt ata ttg gac agc aaa gtg gat tat 480 Val Thr Ala Ile Lys Thr Ile ValIle Leu Asp Ser Lys Val Asp Tyr 145 150 155 160 aga ggt tat caa tcc atggac aac ttt att aaa aaa aac act cca caa 528 Arg Gly Tyr Gln Ser Met AspAsn Phe Ile Lys Lys Asn Thr Pro Gln 165 170 175 ggt ttc aaa gga tca agtttt aaa act gta gaa gtt aac cgc aaa gaa 576 Gly Phe Lys Gly Ser Ser PheLys Thr Val Glu Val Asn Arg Lys Glu 180 185 190 caa gtt gct ctt ata atgaac tct tcg ggt tca acc ggt ttg cca aaa 624 Gln Val Ala Leu Ile Met AsnSer Ser Gly Ser Thr Gly Leu Pro Lys 195 200 205 ggt gtg caa ctt act catgaa aat atc gtc act aga ttt tct cac gct 672 Gly Val Gln Leu Thr His GluAsn Ile Val Thr Arg Phe Ser His Ala 210 215 220 aga gat cca att tat ggaaac caa gtt tca cca ggc acg gct att tta 720 Arg Asp Pro Ile Tyr Gly AsnGln Val Ser Pro Gly Thr Ala Ile Leu 225 230 235 240 act gta gta cca ttccat cat ggt ttt ggt atg ttt act act tta ggc 768 Thr Val Val Pro Phe HisHis Gly Phe Gly Met Phe Thr Thr Leu Gly 245 250 255 tat cta act tgt ggtttt cgt att gtc atg tta acg aaa ttt gac gaa 816 Tyr Leu Thr Cys Gly PheArg Ile Val Met Leu Thr Lys Phe Asp Glu 260 265 270 gag act ttt tta aaaaca ctg caa gat tac aaa tgt tca agc gtt att 864 Glu Thr Phe Leu Lys ThrLeu Gln Asp Tyr Lys Cys Ser Ser Val Ile 275 280 285 ctt gta ccg act ttgttt gca att ctt aat aga agt gaa tta ctc gat 912 Leu Val Pro Thr Leu PheAla Ile Leu Asn Arg Ser Glu Leu Leu Asp 290 295 300 aaa tat gat tta tcaaat tta gtt gaa att gca tct ggc gga gca cct 960 Lys Tyr Asp Leu Ser AsnLeu Val Glu Ile Ala Ser Gly Gly Ala Pro 305 310 315 320 tta tct aaa gaaatt ggt gaa gct gtt gct aga cgt ttt aat tta ccg 1008 Leu Ser Lys Glu IleGly Glu Ala Val Ala Arg Arg Phe Asn Leu Pro 325 330 335 ggt gtt cgt caaggc tat ggt tta aca gaa aca acc tct gca att att 1056 Gly Val Arg Gln GlyTyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Ile 340 345 350 atc aca ccg gaaggc gat gat aaa cca ggt gct tct ggc aaa gtt gtg 1104 Ile Thr Pro Glu GlyAsp Asp Lys Pro Gly Ala Ser Gly Lys Val Val 355 360 365 cca tta ttt aaagca aaa gtt atc gat ctt gat act aaa aaa act ttg 1152 Pro Leu Phe Lys AlaLys Val Ile Asp Leu Asp Thr Lys Lys Thr Leu 370 375 380 ggc ccg aac agacgt gga gaa gtt tgt gta aag ggt cct atg ctt atg 1200 Gly Pro Asn Arg ArgGly Glu Val Cys Val Lys Gly Pro Met Leu Met 385 390 395 400 aaa ggt tatgta gat aat cca gaa gca aca aga gaa atc ata gat gaa 1248 Lys Gly Tyr ValAsp Asn Pro Glu Ala Thr Arg Glu Ile Ile Asp Glu 405 410 415 gaa ggt tggttg cac aca gga gat att ggg tat tac gat gaa gaa aaa 1296 Glu Gly Trp LeuHis Thr Gly Asp Ile Gly Tyr Tyr Asp Glu Glu Lys 420 425 430 cat ttc tttatc gtg gat cgt ttg aag tct tta atc aaa tac aaa gga 1344 His Phe Phe IleVal Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly 435 440 445 tat caa gtacca cct gct gaa tta gaa tct gtt ctt ttg caa cat cca 1392 Tyr Gln Val ProPro Ala Glu Leu Glu Ser Val Leu Leu Gln His Pro 450 455 460 aat att tttgat gcc ggc gtt gct ggc gtt cca gat cct ata gct ggt 1440 Asn Ile Phe AspAla Gly Val Ala Gly Val Pro Asp Pro Ile Ala Gly 465 470 475 480 gag cttccg gga gct gtt gtt gta ctt aag aaa gga aaa tct atg act 1488 Glu Leu ProGly Ala Val Val Val Leu Lys Lys Gly Lys Ser Met Thr 485 490 495 gaa aaagaa gta atg gat tac gtt gct agt caa gtt tca aat gca aaa 1536 Glu Lys GluVal Met Asp Tyr Val Ala Ser Gln Val Ser Asn Ala Lys 500 505 510 cgt ttgcgt ggt ggt gtc cgt ttt gtg gac gaa gta cct aaa ggt ctc 1584 Arg Leu ArgGly Gly Val Arg Phe Val Asp Glu Val Pro Lys Gly Leu 515 520 525 act ggtaaa att gac ggt aaa gca att aga gaa ata ctg aag aaa cca 1632 Thr Gly LysIle Asp Gly Lys Ala Ile Arg Glu Ile Leu Lys Lys Pro 530 535 540 gtt gctaag atg 1644 Val Ala Lys Met 545 6 548 PRT Luciola lateralis 6 Met GluAsn Met Glu Asn Asp Glu Asn Ile Val Tyr Gly Pro Glu Pro 1 5 10 15 PheTyr Pro Ile Glu Glu Gly Ser Ala Gly Ala Gln Leu Arg Lys Tyr 20 25 30 MetAsp Arg Tyr Ala Lys Leu Gly Ala Ile Ala Phe Thr Asn Ala Leu 35 40 45 ThrGly Val Asp Tyr Thr Tyr Ala Glu Tyr Leu Glu Lys Ser Cys Cys 50 55 60 LeuGly Glu Ala Leu Lys Asn Tyr Gly Leu Val Val Asp Gly Arg Ile 65 70 75 80Ala Leu Cys Ser Glu Asn Cys Glu Glu Phe Phe Ile Pro Val Leu Ala 85 90 95Gly Leu Phe Ile Gly Val Gly Val Ala Pro Thr Asn Glu Ile Tyr Thr 100 105110 Leu Arg Glu Leu Val His Ser Leu Gly Ile Ser Lys Pro Thr Ile Val 115120 125 Phe Ser Ser Lys Lys Gly Leu Asp Lys Val Ile Thr Val Gln Lys Thr130 135 140 Val Thr Ala Ile Lys Thr Ile Val Ile Leu Asp Ser Lys Val AspTyr 145 150 155 160 Arg Gly Tyr Gln Ser Met Asp Asn Phe Ile Lys Lys AsnThr Pro Gln 165 170 175 Gly Phe Lys Gly Ser Ser Phe Lys Thr Val Glu ValAsn Arg Lys Glu 180 185 190 Gln Val Ala Leu Ile Met Asn Ser Ser Gly SerThr Gly Leu Pro Lys 195 200 205 Gly Val Gln Leu Thr His Glu Asn Ile ValThr Arg Phe Ser His Ala 210 215 220 Arg Asp Pro Ile Tyr Gly Asn Gln ValSer Pro Gly Thr Ala Ile Leu 225 230 235 240 Thr Val Val Pro Phe His HisGly Phe Gly Met Phe Thr Thr Leu Gly 245 250 255 Tyr Leu Thr Cys Gly PheArg Ile Val Met Leu Thr Lys Phe Asp Glu 260 265 270 Glu Thr Phe Leu LysThr Leu Gln Asp Tyr Lys Cys Ser Ser Val Ile 275 280 285 Leu Val Pro ThrLeu Phe Ala Ile Leu Asn Arg Ser Glu Leu Leu Asp 290 295 300 Lys Tyr AspLeu Ser Asn Leu Val Glu Ile Ala Ser Gly Gly Ala Pro 305 310 315 320 LeuSer Lys Glu Ile Gly Glu Ala Val Ala Arg Arg Phe Asn Leu Pro 325 330 335Gly Val Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Ile 340 345350 Ile Thr Pro Glu Gly Asp Asp Lys Pro Gly Ala Ser Gly Lys Val Val 355360 365 Pro Leu Phe Lys Ala Lys Val Ile Asp Leu Asp Thr Lys Lys Thr Leu370 375 380 Gly Pro Asn Arg Arg Gly Glu Val Cys Val Lys Gly Pro Met LeuMet 385 390 395 400 Lys Gly Tyr Val Asp Asn Pro Glu Ala Thr Arg Glu IleIle Asp Glu 405 410 415 Glu Gly Trp Leu His Thr Gly Asp Ile Gly Tyr TyrAsp Glu Glu Lys 420 425 430 His Phe Phe Ile Val Asp Arg Leu Lys Ser LeuIle Lys Tyr Lys Gly 435 440 445 Tyr Gln Val Pro Pro Ala Glu Leu Glu SerVal Leu Leu Gln His Pro 450 455 460 Asn Ile Phe Asp Ala Gly Val Ala GlyVal Pro Asp Pro Ile Ala Gly 465 470 475 480 Glu Leu Pro Gly Ala Val ValVal Leu Lys Lys Gly Lys Ser Met Thr 485 490 495 Glu Lys Glu Val Met AspTyr Val Ala Ser Gln Val Ser Asn Ala Lys 500 505 510 Arg Leu Arg Gly GlyVal Arg Phe Val Asp Glu Val Pro Lys Gly Leu 515 520 525 Thr Gly Lys IleAsp Gly Lys Ala Ile Arg Glu Ile Leu Lys Lys Pro 530 535 540 Val Ala LysMet 545 7 1644 DNA Luciola lateralis CDS (1)..(1644) 7 atg gaa aac atggag aac gat gaa aat att gtg tat ggt cct gaa cca 48 Met Glu Asn Met GluAsn Asp Glu Asn Ile Val Tyr Gly Pro Glu Pro 1 5 10 15 ttt tac cct attgaa gag gga tct gct gga gca caa ttg cgc aag tat 96 Phe Tyr Pro Ile GluGlu Gly Ser Ala Gly Ala Gln Leu Arg Lys Tyr 20 25 30 atg gat cga tat gcaaaa ctt gga gca att gct ttt act aac gca ctt 144 Met Asp Arg Tyr Ala LysLeu Gly Ala Ile Ala Phe Thr Asn Ala Leu 35 40 45 acc ggt gtc gat tat acgtac gcc gaa tac tta gaa aaa tca tgc tgt 192 Thr Gly Val Asp Tyr Thr TyrAla Glu Tyr Leu Glu Lys Ser Cys Cys 50 55 60 cta gga gag gct tta aag aattat ggt ttg gtt gtt gat gga aga att 240 Leu Gly Glu Ala Leu Lys Asn TyrGly Leu Val Val Asp Gly Arg Ile 65 70 75 80 gcg tta tgc agt gaa aac tgtgaa gaa ttc ttt att cct gta tta gcc 288 Ala Leu Cys Ser Glu Asn Cys GluGlu Phe Phe Ile Pro Val Leu Ala 85 90 95 ggt tta ttt ata ggt gtc ggt gtggct cca act aat gag att tac act 336 Gly Leu Phe Ile Gly Val Gly Val AlaPro Thr Asn Glu Ile Tyr Thr 100 105 110 cta cgt gaa ttg gtt cac agt ttaggc atc tct aag cca aca att gta 384 Leu Arg Glu Leu Val His Ser Leu GlyIle Ser Lys Pro Thr Ile Val 115 120 125 ttt agt tct aaa aaa gga tta gataaa gtt ata act gta caa aaa acg 432 Phe Ser Ser Lys Lys Gly Leu Asp LysVal Ile Thr Val Gln Lys Thr 130 135 140 gta act gct att aaa acc att gttata ttg gac agc aaa gtg gat tat 480 Val Thr Ala Ile Lys Thr Ile Val IleLeu Asp Ser Lys Val Asp Tyr 145 150 155 160 aga ggt tat caa tcc atg gacaac ttt att aaa aaa aac act cca caa 528 Arg Gly Tyr Gln Ser Met Asp AsnPhe Ile Lys Lys Asn Thr Pro Gln 165 170 175 ggt ttc aaa gga tca agt tttaaa act gta gaa gtt aac cgc aaa gaa 576 Gly Phe Lys Gly Ser Ser Phe LysThr Val Glu Val Asn Arg Lys Glu 180 185 190 caa gtt gct ctt ata atg aactct tcg ggt tca acc ggt ttg cca aaa 624 Gln Val Ala Leu Ile Met Asn SerSer Gly Ser Thr Gly Leu Pro Lys 195 200 205 ggt gtg caa ctt act cat gaaaat gca gtc act aga ttt tct cac gct 672 Gly Val Gln Leu Thr His Glu AsnAla Val Thr Arg Phe Ser His Ala 210 215 220 aga gat cca att tat gga aaccaa gtt tca cca ggc acg gct att tta 720 Arg Asp Pro Ile Tyr Gly Asn GlnVal Ser Pro Gly Thr Ala Ile Leu 225 230 235 240 act gta gta cca ttc catcat ggt ttt ggt atg ttt act act tta ggc 768 Thr Val Val Pro Phe His HisGly Phe Gly Met Phe Thr Thr Leu Gly 245 250 255 tat cta act tgt ggt tttcgt att gtc atg tta acg aaa ttt gac gaa 816 Tyr Leu Thr Cys Gly Phe ArgIle Val Met Leu Thr Lys Phe Asp Glu 260 265 270 gag act ttt tta aaa acactg caa gat tac aaa tgt tca agc gtt att 864 Glu Thr Phe Leu Lys Thr LeuGln Asp Tyr Lys Cys Ser Ser Val Ile 275 280 285 ctt gta ccg act ttg tttgca att ctt aat aga agt gaa tta ctc gat 912 Leu Val Pro Thr Leu Phe AlaIle Leu Asn Arg Ser Glu Leu Leu Asp 290 295 300 aaa tat gat tta tca aattta gtt gaa att gca tct ggc gga gca cct 960 Lys Tyr Asp Leu Ser Asn LeuVal Glu Ile Ala Ser Gly Gly Ala Pro 305 310 315 320 tta tct aaa gaa attggt gaa gct gtt gct aga cgt ttt aat tta ccg 1008 Leu Ser Lys Glu Ile GlyGlu Ala Val Ala Arg Arg Phe Asn Leu Pro 325 330 335 ggt gtt cgt caa ggctat ggt tta aca gaa aca acc tct gca att att 1056 Gly Val Arg Gln Gly TyrGly Leu Thr Glu Thr Thr Ser Ala Ile Ile 340 345 350 atc aca ccg gaa ggcgat gat aaa cca ggt gct tct ggc aaa gtt gtg 1104 Ile Thr Pro Glu Gly AspAsp Lys Pro Gly Ala Ser Gly Lys Val Val 355 360 365 cca tta ttt aaa gcaaaa gtt atc gat ctt gat act aaa aaa act ttg 1152 Pro Leu Phe Lys Ala LysVal Ile Asp Leu Asp Thr Lys Lys Thr Leu 370 375 380 ggc ccg aac aga cgtgga gaa gtt tgt gta aag ggt cct atg ctt atg 1200 Gly Pro Asn Arg Arg GlyGlu Val Cys Val Lys Gly Pro Met Leu Met 385 390 395 400 aaa ggt tat gtagat aat cca gaa gca aca aga gaa atc ata gat gaa 1248 Lys Gly Tyr Val AspAsn Pro Glu Ala Thr Arg Glu Ile Ile Asp Glu 405 410 415 gaa ggt tgg ttgcac aca gga gat att ggg tat tac gat gaa gaa aaa 1296 Glu Gly Trp Leu HisThr Gly Asp Ile Gly Tyr Tyr Asp Glu Glu Lys 420 425 430 cat ttc ttt atcgtg gat cgt ttg aag tct tta atc aaa tac aaa gga 1344 His Phe Phe Ile ValAsp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly 435 440 445 tat caa gta ccacct gct gaa tta gaa tct gtt ctt ttg caa cat cca 1392 Tyr Gln Val Pro ProAla Glu Leu Glu Ser Val Leu Leu Gln His Pro 450 455 460 aat att ttt gatgcc ggc gtt gct ggc gtt cca gat cct ata gct ggt 1440 Asn Ile Phe Asp AlaGly Val Ala Gly Val Pro Asp Pro Ile Ala Gly 465 470 475 480 gag ctt ccggga gct gtt gtt gta ctt gaa aaa gga aaa tct atg act 1488 Glu Leu Pro GlyAla Val Val Val Leu Glu Lys Gly Lys Ser Met Thr 485 490 495 gaa aaa gaagta atg gat tac gtt gct agt caa gtt tca aat gca aaa 1536 Glu Lys Glu ValMet Asp Tyr Val Ala Ser Gln Val Ser Asn Ala Lys 500 505 510 cgt ttg cgtggt ggt gtc cgt ttt gtg gac gaa gta cct aaa ggt ctc 1584 Arg Leu Arg GlyGly Val Arg Phe Val Asp Glu Val Pro Lys Gly Leu 515 520 525 act ggt aaaatt gac ggt aaa gca att aga gaa ata ctg aag aaa cca 1632 Thr Gly Lys IleAsp Gly Lys Ala Ile Arg Glu Ile Leu Lys Lys Pro 530 535 540 gtt gct aagatg 1644 Val Ala Lys Met 545 8 548 PRT Luciola lateralis 8 Met Glu AsnMet Glu Asn Asp Glu Asn Ile Val Tyr Gly Pro Glu Pro 1 5 10 15 Phe TyrPro Ile Glu Glu Gly Ser Ala Gly Ala Gln Leu Arg Lys Tyr 20 25 30 Met AspArg Tyr Ala Lys Leu Gly Ala Ile Ala Phe Thr Asn Ala Leu 35 40 45 Thr GlyVal Asp Tyr Thr Tyr Ala Glu Tyr Leu Glu Lys Ser Cys Cys 50 55 60 Leu GlyGlu Ala Leu Lys Asn Tyr Gly Leu Val Val Asp Gly Arg Ile 65 70 75 80 AlaLeu Cys Ser Glu Asn Cys Glu Glu Phe Phe Ile Pro Val Leu Ala 85 90 95 GlyLeu Phe Ile Gly Val Gly Val Ala Pro Thr Asn Glu Ile Tyr Thr 100 105 110Leu Arg Glu Leu Val His Ser Leu Gly Ile Ser Lys Pro Thr Ile Val 115 120125 Phe Ser Ser Lys Lys Gly Leu Asp Lys Val Ile Thr Val Gln Lys Thr 130135 140 Val Thr Ala Ile Lys Thr Ile Val Ile Leu Asp Ser Lys Val Asp Tyr145 150 155 160 Arg Gly Tyr Gln Ser Met Asp Asn Phe Ile Lys Lys Asn ThrPro Gln 165 170 175 Gly Phe Lys Gly Ser Ser Phe Lys Thr Val Glu Val AsnArg Lys Glu 180 185 190 Gln Val Ala Leu Ile Met Asn Ser Ser Gly Ser ThrGly Leu Pro Lys 195 200 205 Gly Val Gln Leu Thr His Glu Asn Ala Val ThrArg Phe Ser His Ala 210 215 220 Arg Asp Pro Ile Tyr Gly Asn Gln Val SerPro Gly Thr Ala Ile Leu 225 230 235 240 Thr Val Val Pro Phe His His GlyPhe Gly Met Phe Thr Thr Leu Gly 245 250 255 Tyr Leu Thr Cys Gly Phe ArgIle Val Met Leu Thr Lys Phe Asp Glu 260 265 270 Glu Thr Phe Leu Lys ThrLeu Gln Asp Tyr Lys Cys Ser Ser Val Ile 275 280 285 Leu Val Pro Thr LeuPhe Ala Ile Leu Asn Arg Ser Glu Leu Leu Asp 290 295 300 Lys Tyr Asp LeuSer Asn Leu Val Glu Ile Ala Ser Gly Gly Ala Pro 305 310 315 320 Leu SerLys Glu Ile Gly Glu Ala Val Ala Arg Arg Phe Asn Leu Pro 325 330 335 GlyVal Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Ile 340 345 350Ile Thr Pro Glu Gly Asp Asp Lys Pro Gly Ala Ser Gly Lys Val Val 355 360365 Pro Leu Phe Lys Ala Lys Val Ile Asp Leu Asp Thr Lys Lys Thr Leu 370375 380 Gly Pro Asn Arg Arg Gly Glu Val Cys Val Lys Gly Pro Met Leu Met385 390 395 400 Lys Gly Tyr Val Asp Asn Pro Glu Ala Thr Arg Glu Ile IleAsp Glu 405 410 415 Glu Gly Trp Leu His Thr Gly Asp Ile Gly Tyr Tyr AspGlu Glu Lys 420 425 430 His Phe Phe Ile Val Asp Arg Leu Lys Ser Leu IleLys Tyr Lys Gly 435 440 445 Tyr Gln Val Pro Pro Ala Glu Leu Glu Ser ValLeu Leu Gln His Pro 450 455 460 Asn Ile Phe Asp Ala Gly Val Ala Gly ValPro Asp Pro Ile Ala Gly 465 470 475 480 Glu Leu Pro Gly Ala Val Val ValLeu Glu Lys Gly Lys Ser Met Thr 485 490 495 Glu Lys Glu Val Met Asp TyrVal Ala Ser Gln Val Ser Asn Ala Lys 500 505 510 Arg Leu Arg Gly Gly ValArg Phe Val Asp Glu Val Pro Lys Gly Leu 515 520 525 Thr Gly Lys Ile AspGly Lys Ala Ile Arg Glu Ile Leu Lys Lys Pro 530 535 540 Val Ala Lys Met545

1-13. (Cancelled)
 14. A luciferase having resistance to a surfactant,wherein said luciferase retains at least 85% of its activity in thepresence of 0.1% surfactant and wherein said luciferase has at least oneamino acid mutation compared to the native luciferase amino acidsequence.
 15. The luciferase of claim 14, wherein the surfactant is acationic surfactant.
 16. The luciferase of claim 14, wherein thesurfactant is a quaternary ammonium salt.
 17. The luciferase of claim14, wherein the surfactant is a benzalkonium chloride.
 18. Theluciferase of claim 14, which is a luciferase derived from the orderCleoptera.
 19. The luciferase of claim 14, which is derived from thefamily firefly.
 20. The luciferase of claim 14, which is derived fromthe family Pyrophorus.
 21. The luciferase of claim 14, which is derivedfrom GENJI firefly, HEIKE firefly, North American firefly or Russianfirefly.
 22. The luciferase of claim 14, which is derived fromPynophorous plagiophthalamus, Arachnocampa luminosa or Rail worm. 23.The luciferase of claim 14, which comprises at least one substitution,deletion or insertion mutation in the amino acid sequence of the nativeluciferase.
 24. The luciferase of claim 14, which comprises at least onemutation at the position corresponding to position 490 of the GENJI orHEIKE firefly luciferase.
 25. An isolated nucleic acid encoding theluciferase of claim
 14. 26. A recombinant DNA comprising the isolatednucleic acid of claim
 25. 27. A recombinant cell comprising therecombinant DNA of claim
 26. 28. A method for making a luciferase whichis surfactant resistant comprising: introducing at least one mutationinto the nucleic acid sequence encoding a luciferase to obtain a nucleicacid encoding a mutant luciferase, expressing said nucleic acid encodingthe mutant luciferase, and selecting a mutant nucleic acid sequencewhich encodes a mutant luciferase which retains at least 85% of itsactivity in the presence of 0.1% surfactant.
 29. The method of claim 28,wherein said surfactant is a cationic surfactant.
 30. The method ofclaim 28, wherein said surfactant is a quaternary ammonium salt.
 31. Themethod of claim 28, wherein said surfactant is benzalkonium chloride.32. A surfactant resistant luciferase produced by the method of claim28.
 33. A method for measuring the intracellular ATP comprising:extracting ATP from a cell sample in the presence of a surfactant,adding a luminescence reagent containing the surfactant resistantluciferase of claim 14 for a time and under conditions suitable toproduce the emission of light, and detecting or measuring the emissionof light.
 34. A method for measuring the intracellular ATP comprising:extracting ATP from a cell sample in the presence of a surfactant,adding a luminescence reagent containing the surfactant resistantluciferase of claim 32 for a time and under conditions suitable toproduce the emission of light, and detecting or measuring the emissionof light.